高脂饮食代谢模型（C57BL/6背景）

小鼠腹腔注射胰岛素后的耐量试验。禁食 4–6 小时；按 1–1.2 U/kg 给予胰岛素；负荷前及负荷后 15、30、60、90、120 分钟测定血糖。不同组别与样本量按具体研究设计确定，本方案不预设实验分组。

基线与负荷后 15、30、60、90、120 分钟血糖时间序列；可计算血糖下降百分比或曲线下面积。

340 nm吸光度变化对应的葡萄糖浓度。

Fasting Fasting begins first thing in the morning (around 9am). Fast mice for 4-6 hours before ITT begins. Transfer mice to clean cage and wire top. Keep water bottles during the fasting period. 1. If using High Fat diet (HFD), save food to give back at the end of the ITT. 1. Mark tails with a sharpie for easier identification during ITT. Insulin Solution prep Stock 1: 50ul of 100U/ml Humulin R into 4950ul of cold sterile 1X PBS. Invert solution 3-4 times to mix. Stock 2: 1ml of Stock 1 into 9ml of cold sterile 1X PBS. Invert solution 3-4 times to mix. Keep on ice until use ITT Weigh mouse to obtain body weight for dose calculations. Calculate bolus dose of insulin (**1-1.2 units/kg**). Weight of mouse (g) x 10 = ul insulin stock 2 solution ITT - template spreadsheet.xlsx ITT Prepare syringes using 1cc syringe and 26G needle. Load all syringes with calculated insulin dose and position in front of home cage. ITT Prior to the delivery of the insulin solution, a time “zero” blood glucose level must be measured. Restrain mouse and clean tail with 70% ethanol/gauze. Extract a small amount of blood from the tail vein onto One Touch Ultra blood glucose strips used with the One Touch Ultra 2 blood glucose monitoring system. ITT Administer the correct dose of Insulin solution using an IP (intraperitoneal) injection method. - Restrain the mouse. Then place the mouse is in dorsal recumbency with the head down allowing the viscera to move cranially. - Using the bend of the knee and the ventral abdominal midline as landmarks, the needle is inserted half way between the midline and lateral side of the animal at the natural bend of the knee. Insert the needle at a 30 to 45 degree angle into either the left or right lower abdominal quadrant. - Prior to injection, aspirate to make sure the needle has not penetrated a blood vessel, the intestines or the urinary bladder. When aspirating the syringe, you should see an air bubble in the hub of the needle, and not any form of fluid. - Inject and return mouse to cage. ITT Begin timing from the point of successfully delivering the insulin solution. ITT Test and record the blood glucose level (see step 5) at times 15, 30, 60, 90, and 120 minutes after the initial glucose delivery. Record each value on Insulin Tolerance Test record sheet (template found on step 2). Record condition of animal throughout and after procedure on the Insulin Tolerance Test recored sheet. ITT Return food and environmental enrichments to cages.

Calibrate Cobas for Glucose analysis by running a multi analyte standard and two control serum. Calibrate Cobas for Glucose analysis by running a multi analyte standard and two control serum. Sample Handling as performed by the Cobas Mira Plus. Sample Handling as performed by the Cobas Mira Plus. a) Pipette 3 µL of sample into cuvette. b) Absorbance is measured at 340 nm. c) Add 100 µL of Glucose liquid reagent. d) Mixture is incubated at 37ºC for 10 minutes. e) Absorbance is measured at 340 nm. Change in absorbance is calculated.